Luciferases are enzymes that catalyze the oxidation of a substrate (e.g., luciferin or coelenterazine) with the concomitant release of photons of light. Luciferases have been isolated from numerous species, including Coleopteran arthropods and many sea creatures as well as bacteria. Because it is easily detectable and its activity can be quantified with high precision, luciferases have been widely used to study gene expression and protein localization. Unlike green fluorescent protein (GFP), which requires up to 30 minutes to form its chromophore, the products of luciferases can be detected immediately upon completion of synthesis of the polypeptide chain (if substrate and oxygen are also present). In addition, no post-translational modifications are required for enzymatic activity, and the enzyme contains no prosthetic groups, bound cofactors, or disulfide bonds. Luciferases are useful reporters in numerous species and in a wide variety of cells.
Luciferases possess additional features that render them particularly useful as reporter molecules for biosensing, i.e., molecules which reveal molecular properties of a system. Most catalytic reactions generate less than the energy of hydrolysis for two molecules of ATP, or about 70 kJ/mole. However, the luminescence elicited by luciferases has much higher energy content. For instance, the reaction catalyzed by firefly luciferase (560 nm) emits 214 kJ/mole of energy. Furthermore, luciferases are also highly efficient at converting chemical energy into photons, i.e., they have high quantum yields. Luciferases are thus extremely efficient for generating detectable signals.
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are effector lymphocytes that share common cytotoxic pathways that are necessary for defense against virus-infected or transformed cells such as tumor cells. NK cells play a major role in the rejection of tumors and cells infected by viruses. They kill cells by releasing, via exocytosis, into target cells small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis, i.e., programmed cell death. The granule-exocytosis pathway powerfully activates cell-death pathways that operate through the activation of apoptotic cysteine proteases (caspases), but it also leads to cell death in the absence of activated caspases. Perforin and granzymes induce target-cell apoptosis cooperatively. Granzymes, a family of structurally related serine proteases, are necessary for triggering apoptosis in target cells, but they depend on being appropriately delivered by perforin.
Granzyme B and granzyme B isoforms, which cleave target-cell proteins at specific aspartate residues, is a potent activator of caspase-mediated, as well as caspase-independent cell death. The uptake of granzyme B into target cells is believed to be mediated (at least in part) by endocytosis through the cation-independent mannose 6-phosphate receptor. Granzyme B mainly triggers caspase activation indirectly rather than by direct caspase processing. It achieves this by directly activating pro-apoptotic ‘BH3-only’ members of the BCL-2 family, such as BH3-interacting domain death agonist (BID), which results in the leakage of pro-apoptotic mitochondrial mediators, such as cytochrome c, into the cytosol. BID-independent pathways for granzyme-mediated apoptosis also seem to exist. Some viruses encode potent granzyme-B inhibitors, and the endogenous granzyme-B inhibitor PI9 might be expressed aberrantly by some cancer cells.
CTLs and NK cells are critical effector cells of the immune system. The components of NK function include: NK numbers, granule number/content, immune synapse formation, polarization toward synapse, docking to membrane, priming, fusion, and delivery of granzyme. Measurement of target cell damage has historically been an important measure of CTL and NK cell function. The gold standard for CTL-mediated cytotoxicity has been the chromium release assay (“[51Cr] release assay”). The [51Cr] release assay involves the labeling of viable cells with [51Cr] which binds tightly to most intracellular proteins. After washing, the cells are incubated with a cytotoxic agent, e.g., effector cells. Labeled proteins are released into the cell media due to damage and/or leakage of the cell membrane caused by the triggering of cell death by the cytotoxic agent. Radioactivity is detected, thereby indicating cell death. As the [51Cr] release assay involves the use of radioactivity, and is time intensive, there exists a need to provide a non-radioactive, sensitive assay for the measurement of cell-mediated cytotoxicity.